LOCUS       Exported                6618 bp ds-DNA     circular SYN 16-JUN-2022
DEFINITION  Mammalian expression of human SIRT3 with flag tag.
ACCESSION   .
VERSION     .
KEYWORDS    SIRT3 Flag
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6618)
  AUTHORS   North BJ, Marshall BL, Borra MT, Denu JM, Verdin E
  TITLE     The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin 
            deacetylase.
  JOURNAL   Mol Cell. 2003 Feb . 11(2):437-44.
  PUBMED    12620231
REFERENCE   2  (bases 1 to 6618)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 16, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6618
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(11..28)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(941..1801)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1981..2000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2172..2551
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2552..2755
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2706..2726
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2800..2819
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2800..2818
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4088..4111
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4146..4163)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4152..4376
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4422..4850
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4509..4528)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4719..4740
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4859..4879)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4864..5193
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5044..5179
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5106..5125
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5260..6054
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5314..5333)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5924..5943
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6228..6349
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6265..6284)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6319..6338
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6398..6414)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6398..6414)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6411..6433)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6422..6438
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(6446..6476)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6491..6512
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
     1981 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     2041 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     2101 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     2161 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     2221 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     2281 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     2341 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     2401 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     2461 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     2521 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     2581 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     2641 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     2701 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     2761 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     2821 gacccaagct ggctagcgtt taaacttaag cttggtaccg agctcggatc cgaattccat
     2881 ggcgttctgg ggttggcgcg ccgcggcagc cctccggctg tggggccggg tagttgaacg
     2941 ggtcgaggcc gggggaggcg tggggccgtt tcaggcctgc ggctgtcggc tggtgcttgg
     3001 cggcagggac gatgtgagtg cggggctgag aggcagccat ggggcccgcg gtgagccctt
     3061 ggacccggcg cgccccttgc agaggcctcc cagacccgag gtgcccaggg cattccggag
     3121 gcagccgagg gcagcagctc ccagtttctt cttttcgagt attaaaggtg gaagaaggtc
     3181 catatctttt tctgtgggtg cttcaagtgt tgttggaagt ggaggcagca gtgacaaggg
     3241 gaagctttcc ctgcaggatg tagctgagct gattcgggcc agagcctgcc agagggtggt
     3301 ggtcatggtg ggggccggca tcagcacacc cagtggcatt ccagacttca gatcgccggg
     3361 gagtggcctg tacagcaacc tccagcagta cgatctcccg taccccgagg ccatttttga
     3421 actcccattc ttctttcaca accccaagcc ctttttcact ttggccaagg agctgtaccc
     3481 tggaaactac aagcccaacg tcactcacta ctttctccgg ctgcttcatg acaaggggct
     3541 gcttctgcgg ctctacacgc agaacatcga tgggcttgag agagtgtcgg gcatccctgc
     3601 ctcaaagctg gttgaagctc atggaacctt tgcctctgcc acctgcacag tctgccaaag
     3661 acccttccca ggggaggaca ttcgggctga cgtgatggca gacagggttc cccgctgccc
     3721 ggtctgcacc ggcgttgtga agcccgacat tgtgttcttt ggggagccgc tgccccagag
     3781 gttcttgctg catgtggttg atttccccat ggcagatctg ctgctcatcc ttgggacctc
     3841 cctggaggtg gagccttttg ccagcttgac cgaggccgtg cggagctcag ttccccgact
     3901 gctcatcaac cgggacttgg tggggccctt ggcttggcat cctcgcagca gggacgtggc
     3961 ccagctgggg gacgtggttc acggcgtgga aagcctagtg gagcttctgg gctggacaga
     4021 agagatgcgg gaccttgtgc agcgggaaac tgggaagctt gatggaccag acaaagaatt
     4081 cggaggagac tacaaggacg acgatgacaa gtaatctaga gggcccgttt aaacccgctg
     4141 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     4201 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     4261 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4321 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     4381 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     4441 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4501 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4561 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4621 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4681 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4741 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4801 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     4861 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4921 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4981 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     5041 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     5101 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     5161 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     5221 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     5281 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     5341 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     5401 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     5461 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     5521 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     5581 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     5641 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     5701 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     5761 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     5821 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     5881 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     5941 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     6001 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     6061 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     6121 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     6181 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     6241 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     6301 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     6361 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     6421 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     6481 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     6541 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6601 gtttgcgtat tgggcgct
//
